Kupffer cells are one type of liver-specific resident tissue macrophages, and are cells playing important biological defense functions such as recognition of foreign materials and induction of immune responses. Moreover, Kupffer cells are closely associated with induction and onset of liver dysfunction, hepatitis, cirrhosis, and the like. Thus, Kupffer cells are considered to be a very important target for elucidating the liver disease state, developing therapeutic drugs, and so forth. For these reasons, many attempts have been made so far to isolate Kupffer cells from livers, and to proliferate isolated Kupffer cells by culturing in vitro.
As a result, as the method for isolating Kupffer cells, methods have been established in which: a cell suspension obtained through liver perfusion with a digestive enzyme such as a collagenase and cell dispersion is centrifuged at a low speed to remove hepatic parenchymal cells, and a Kupffer cell fraction is harvested from non-parenchymal cells in a supernatant on the basis of the cell density using an elutriation centrifuge (Non Patent Literatures 1 to 3). However, the isolation of Kupffer cells has problems to be solved because it requires special equipment, and furthermore complicated processes dependent on skilled techniques. Moreover, each time Kupffer cells are isolated, an animal has to be sacrificed to isolate Kupffer cells from the liver thereof. This makes it difficult to obtain a large amount of Kupffer cells conveniently.
Meanwhile, there is known a method for separating Kupffer cells from liver cells by utilizing the adherence of the Kupffer cells to a plastic dish (Non Patent Literature 4). However, this method also has difficulty obtaining a large amount of Kupffer cells conveniently because the amount of Kupffer cells obtained depends on the amount of liver that is the starting material. In addition, the proliferation ability of Kupffer cells is known to be low. Even if Kupffer cells can be separated by this method, it is still difficult to produce a large amount of Kupffer cells conveniently.
Further, there is known a method in which cells derived from the liver are subjected to limiting dilution and cultured in vitro in the presence of various cytokines and the like to obtain Kupffer cell clones (Patent Literature 1). However, this method requires culturing for quite a long period such as 120 days to obtain a large amount of Kupffer cells. In addition, since the proliferation ability of Kupffer cells is known to be low, Kupffer cells alone do not proliferate. Accordingly, even if Kupffer cell clones can be obtained by this method, it is still difficult to efficiently produce a large amount of Kupffer cells in a convenient way.